The immune system is regulated in part by a complex network of chemical signals. These signals include the interleukins such as IL-1xcex1 and IL-1xcex2. IL-1xcex2 is a polypeptide hormone synthesized and secreted by stimulated monocytes. The initial translation product of IL-1xcex2 is a 31 kDa precursor polypeptide having relatively low biological activity. After synthesis, the 31 kDa precursor for IL-1xcex2 is enzymatically cleaved to its highly active mature form which has a size of about 17.5 kDa. The N-terminus of mature IL-1xcex2 derived from human activated monocytes has been characterized by an N-terminal amino acid sequence beginning with Ala-Pro. The N-terminal Ala residue of human mature IL-1xcex2 is in the 117 position and an Asp residue is in the 116 position counting from the N-terminus of human precursor IL-1 xcex2 polypeptide. Mature IL-1xcex2 consists of the C-terminal 153 residues of the precursor polypeptide.
Many physiological actions and biological activities of IL-1xcex2 have been identified. IL-1xcex2 biological activity is often determined by assaying for stimulation of thymocyte proliferation. IL-1xcex2 activities also include stimulation of B-lymphocyte maturation, lymphocyte proliferation, stimulation of fibroblast growth and induction of acute-phase protein synthesis by hepatocytes.
Other biological activities have been attributed to IL-1xcex2 polypeptides. These include control of differentiation and activation of lymphocytes, stimulation of lymphokine and prostaglandin production, promotion of inflammation, induction of acute phase proteins, stimulation of bone resorption, and alteration of the level of iron and zinc in blood. Moreover, it has been found that IL-1xcex2 can stimulate the hypothalamus-pituitary-adrenal axis, suggesting that IL-1xcex2 is integrated in the complex neuroendocrine network that controls homeostasis.
Maturation and release of mature IL-1xcex2 from macrophages does not proceed by conventional means normally associated with most secretory proteins because the precursor IL-1xcex2 polypeptide lacks a hydrophobic signal sequence. Further, IL-1xcex2 is not associated with a membrane-bound compartment in monocytes. Most secretory proteins are characterized by the presence of a hydrophobic stretch of amino acids called a signal sequence. The signal sequence directs the translocation of the protein across the membrane of the endoplasmic reticulum during protein synthesis. The protein is subsequently ushered out of the cell via exocytosis. Most secreted proteins have a signal sequence at the amino terminal that is removed upon translocation. Other proteins, such as ovalbumin, have an internal signal sequence that is not removed upon translocation. The precursor form of IL-1xcex2 lacks any region (either amino terminal or internal) with sufficient hydrophobicity and length to qualify as a signal sequence.
It is very likely that the biological activity of IL-1xcex2 is tightly linked to the structural integrity of the protein molecule, for deletion of amino acids from the mature protein is accompanied by severe diminution of bioactivity. Several groups have introduced point mutations in an attempt to probe receptor ligand interactions (Jobling et al., 1988; Dechiara, T. M. et al. [1986] Proc. Natl. Acad. Sci. USA 83:8303-8307; Mosley, B., S. K. Dower, S. Gillis, D. Cosman [1987] Proc. Natl. Acad Sci. USA 84:4572-4576). Huang et al. (Huang, J. J. et al. [1987] FEBS Letters 223:294-298) reported that the biological activity of IL-1 xcex2 was increased four- to seven-fold by changing the native NH2-terminal sequence from ala-pro-val-arg-serto thr-met-val-arg-ser; however, further alteration of arginine120 to generate thr-met-val-glu-ser effectively abolished bioactivity. Circular dichroism data demonstrated no major structural differences among the proteins. Gronenborn et al. (Gronenborn, A. M., P. T. Wingfield, H. F. McDonald, U. Schmeissner, G.M. Clore [1988] FEBS Letters 231:135-138) mutated IL-1-alpha histidine and tryptophan residues without effect upon receptor binding affinity, while MacDonald et al. (MacDonald, H. R. et al. [1986] FEBS Letters 209:295-298) reported IL-1 histidine muteins with 2-100 fold less competitive binding activity than the wild-type protein.
Excessive or unregulated IL-1 has been implicated in various diseases. These include rheumatoid arthritis (see, e.g., Fontana et al. [1982] Arthritis Rheum. 22:49-53); osteoarthritis (see, e.g., Wood et al. [1983] Arthritis Rheum. 26:975); toxic shock syndrome (see, e.g., Ikejima and Dinarello [1985] J. Leukocyte Biology 37:714); other acute or chronic inflammatory disease states such as the inflammatory reaction induced by endotoxin (see, e.g., Habicht and Beck [1985] J. Leukocyte Biology 37:709); and other chronic inflammatory disease states such as tuberculosis (see, e.g., Chesque et al. [1985] J. Leukocyte Biology 37:690). Benjamin et al. ([1985] xe2x80x9cAnnual Reports in Medicinal Chemistryxe2x80x9420,xe2x80x9d Chapter 18, pp. 173-183, Academic Press, Inc.) disclose that excessive IL-1 production is implicated in psoriatic arthritis, Reiter""s syndrome, rheumatoid arthritis, osteoarthritis, gout, traumatic arthritis, rubella arthritis, and acute synovitis.
Dinarello ([1985] J. Clinical Immunol. 5(5):287-297)reviews the biological activities which have been attributed to IL-1.
Another form of IL-1, the naturally-occurring receptor antagonist protein (IL-1ra) (Carter, D. B., M. R. Deibel, C. J. Dunn et aL [1990] Nature 344:633-638; Hannum, C. H., C. J. Wilcox, W. P. Arend et al. [1990] Nature 343:336-340; Seckinger, P. K. Williamson, J.-F. Balavoine et al. [1987] J. Immunol. 139:1541-1545) may play an important role in modulating the effects of IL-1. Interleukin-1 biological activity is initiated by interaction with either the type I or type II cellular IL-1 receptors (Dower, S. K., S. R. Kronheim, C. J. March et al. [1984] J. Exp. Med. 162:501; Sims, J. E., S. K. Dower [1990] Year in Immunology 6:112-126; Sims, J. E., C. J. March, D. Cosman et al. [1988] Science 241:585-588; Spriggs, M. K., P. J. Lioubin, J. Slack et al. [1990] J. Biol. Chem. 265:22499-22505), each of which possesses three immunoglobulin-fold extracellular ligand-binding domains. The IL-1ra also binds to IL-1 receptors, but this protein has not been reported to elicit biological responses (Dripps, D. J., B. J. Brandhuber, R. C. Thompson, S. P. Eisenberg [1991] J. Biol. Chem. 266:10331-10336). The three-dimensional structures of both IL-1xcex1 and IL-1xcex2 have been reported (Clore, G. M., P. T. Wingfield, A. M. Gronenbom [1991] Biochemistry 30:2315-2323; Driscoll, P. C., G. M. Clore, D. Marion et al. [1990] Biochemistry 29:3542-3556; Finzel et al., supra; Graves et al. [1990],supra; Priestle et al. [1988], supra; Priestle, J. P., Schar, H. P., M. G. Grutter [1990] Cytokines and Lipocortins 349:297-307) and both receptor types have been molecularly cloned and expressed (McMahan, C. J., J. L. Slack, B. Mosely et al. [1991] EMBO J. 10:2821; Sims et al. [1988], supra; Spriggs et al., supra).
By changing the R127 in xcex2-strand 1 of the native IL-1xcex2 to a glycine, it has been determined that the receptor binding and biological activity domains of the protein are at least partially distinct. Others have subsequently reported similar effects resulting from mutating other amino acids located in the hydrogen-bonded antiparallel xcex2-strand 1 and 12 of IL-1xcex2 (Ju, G., E. Labriolatompkins, C. A. Campen et al. [1991] Proc. Natl. Acad. Sci. USA 88:2658-2662; Young, P., V. Kumar, J. Lillquist et al. [1990] Lymphokine Res. 9:599). Ju et al., supra, also showed that the IL-1ra protein can be converted to a partial agonist by mutating a residue in a similar structural location.
It is known that in many cases that both cellular and/or humoral immune responses to an antigen administered to an animal can be enhanced or increased by immunizing the animal with the antigen in conjunction with some type of adjuvant. An adjuvant, in broad terms, may be thought of as a compound or composition which can enhance or amplify an animal""s immune response (e.g., an increase in antibody titer) to an antigen or immunogen. Various adjuvants are known in the art, including Freund""s (complete and incomplete), muramyl dipeptide (MDP), and alum.
Mucosal immunization typically induces a state of immunologic unresponsiveness known as xe2x80x9coral tolerancexe2x80x9d or more correctly mucosally induced tolerance. The induction of the appropriate mucosal immune response to human pathogens (HIV, HSV, measles, mumps, RSV, etc.) may provide superior systemic immune responses for protection against infection. Mucosal immune responses have been reported to prevent infection while systemic immune responses usually resolve the infection (they do not prevent infection).
The most potent and best-studied mucosal adjuvant is cholera toxin (CT, Elson et al. [1994] in Handbook of Mucosal Immunology, p 391). However, CT is likely unsafe for use as a mucosal adjuvant in humans because as little as 5 micrograms (xcexcg) of CT causes massive diarrhea when intragastrically administered to human volunteers (Levine et al. [1983] Microbiological Reviews 47:510). Moreover, in some cases the use of CT as a mucosal adjuvant in research animals has been associated with the production of antigen-specific IgE responses and lethal anaphylactic reactions. See, e.g., Snider et al. [1994] J. Immunol 153:647; Marinaro et al. [1995] J. Immunol 155:4621.
To repress the toxicity associated with toxin adjuvants, mutant CT, LT, and PT molecules have been produced that exhibit reduced or undetectable toxic activity while maintaining mucosal adjuvant activity (O""Hagan [1997] Journal of Pharmacy and Pharmacology 49:1). Although these molecules possess potent adjuvant activity with reduced toxicity, they maintain immunogenic properties when administered to experimental animals. See e.g. Douce et al. [1977] Infection and Immunity 65:2821. Thus, the immunogenicity of these mutant toxin molecules also prevents their widespread and repeated use as mucosal adjuvants in that pre-existing immunity to CT reduces their adjuvant activity (Wu et al. [1994] Vaccine 12:215).
PCT Publication No. WO 91/01143 to Pillai et al. describes interluekin (IL)-containing vaccine compositions which comprise a mixture of antigen and an adjuvant amount of an IL adsorbed onto a mineral in suspension, and a preservative. The mineral is described as preferably being alum. Alum is described as stabilizing the biological activity of the IL. Exemplary IL""s includes IL-1xcex1, IL-1xcex2, IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7. It is noted that in the absence of alum, IL""s have short half lives. Carbohydrate-containing units found uniquely on cancer cells or found associated with allergens are particularly described antigens. However, the problems associated with oral or mucosally-induced tolerance of antigens are not addressed.
U.S. Pat. No. 5,334,379 issued to Pillai et al. on Aug. 2, 1994 describes cytokine and hormone carriers for conjugate vaccines. The antigens described in this patent are bound or genetically fused with a cytokine, lymphokine, hormone or growth factor having immunomodulating activity. Particularly contemplated antigens include the carbohydrate-containing antigens often present in bacteria. Exemplary cytokines, lumphokines and hormones include IL-1xcex1, IL-1xcex2, IL-2, prolactin, EGF, TGF, GM-CSF, GCSF, IGF-1, somatotropin, or insulin. The conjugates may be prepared by any of the known complex, biologically compatible methods for coupling carbohydrate-containing antigens or other antigens to carriers. Covalent coupling is described as a preferred method. Indeed, Examples 1 and 2 describe the elaborate reactions required to conjugate or bind the antigens to the cytokine, lumphokine, hormone or growth factor. However, the problems associated with oral or mucosally-induced tolerance of antigens are not addressed.
A recent review article by Kramer et al. (Kramer et al. [1995] entitled xe2x80x9cCytokine Mediated Effects in Mucosal Immunologyxe2x80x9d in Immunology and Cell Biology 73:389) discusses the role of IL-5, IL-6, and TGF-xcex2 in the induction of mucosal IgA responses. Particularly, this paper discussed published results from experiments done in mice lacking a functional IL-5 or IL-6 gene. It also discusses papers which describe the co-expression of IL-5 or IL-6 with vaccine antigen in a live vaccinia virus, and which describe that the co-expression of IL-5 or IL-6 enhances mucosal IgA responses. However, the article then suggests that complex delivery methods will be required to deliver the cytokines to the mucosa.
Another recent review article by O""Hagan entitled xe2x80x9cRecent Advances in Vaccine Adjuvants for Systemic and Mucosal Adjuvantsxe2x80x9d in the Journal of Pharmacy and Pharmacology 49:1 (1997) discusses the state of the use of adjuvants for systemic and mucosal administration. This review article discusses a number of different adjuvants for use with mucosally administered vaccines including particulates (i.e. microspheres), oil-in-water emulsions, and mutated forms of heat-labile enterotoxin (LT) and cholera toxin (CT). But, this article does not mention the use of cytokines as mucosal vaccine adjuvants.
A journal article by Lin et al. entitled xe2x80x9cPresent Status of the Use of Cytokines as Adjuvants with Vaccines to Protect Against Infectious Diseasesxe2x80x9d in Clinical Infectious Diseases 21:1439 (1995) discusses the use of select cytokines (IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, and IL-12; tumor necrosis factor (TNF); interferon; and GM-CSF as adjuvants. But, the use of cytokines as mucosal vaccine adjuvants is not suggested in this article.
A journal article by Nash et al. entitled xe2x80x9cRecombinant Cytokines as 20 Immunological Adjuvantsxe2x80x9d, Immunology and Cell Biology 71:367 (1993) discusses the use of recombinant ovine IL-2, IL-1xcex1 and tumor necrosis factor-xcex1 (TNF-xcex1) as adjuvants. The formulation of IL-1xcex1 with aluminum hydroxide (alum) is mentioned for use as an adjuvant capable of enhancing secondary humoral responses. But, there is no suggestion of mucosal administration of IL-1xcex1.
More recently, polypeptides and small peptides have been used as adjuvants. U.S. Pat. No. 5,503,841 discloses the use of interleukin-2 (IL-2) as an adjuvant with vaccines. U.S. Pat. No. 5,206,014 discloses the use of a peptide fragment of human IL-1xcex2 as an adjuvant with antigens having low immunogenicity. However, systemic administration of immunomodulators, such as interleukins, as adjuvants can result in an overstimulation or dysfunctional activation of the immune system of the animal. Systemic in vivo administration of IL-1xcex2 has been associated with unwanted side effects, including fever and nausea.
There has been a report that recombinant ovine interleukin-1xcex2 has adjuvant activity when administered intramuscularly to mice or sheep (Vaccine 13:1277-1287). However, this study did not address the use of IL-1 as a mucosal adjuvant. It has also been reported that Interleukin 1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens (Staats, H. F. and F. A. Ennis, Jr. [1999] J. of Immunology 162:6141-6147). Presently, the only mucosal adjuvants that have worked well include bacterial toxins such as cholera toxin, heat-labile toxin, or pertussis toxin. A number of groups are genetically modifying the bacterial toxins so that they maintain adjuvant activity in the absence of toxin activity. However, a foreign protein must still be used as an adjuvant.
There remains a need in the art for adjuvants that stimulate or activate appropriate cells of the immune system without overstimulation or dysfunction.
The present invention pertains to compositions and methods useful as vaccine adjuvants. In a preferred embodiment, the subject invention provides adjuvant compositions comprising a human IL-1xcex2 mutein. More specifically, the adjuvants of the subject invention preferably comprise IL-1xcex2 muteins wherein a positively charged residue (arginine or lysine) is replaced with any of the other 17 natural amino acids.
The IL-1xcex2 vaccine compositions preferably further comprise an antigen, such as whole inactivated or attenuated virus, recombinant or synthetic peptides, and/or other antigenic materials. These IL-1xcex2 muteins have reduced biological activity, as compared to the parent molecule, without loss of receptor binding affinity.
The present invention also pertains to the use of IL-1 muteins for use as a means to increase immune responses in a patient. In an exemplified embodiment of the present invention, muteins of IL-1xcex2 can be used as an immunomodulator for increasing immune responses.
In accordance with the present invention, a method of eliciting an immune response against an antigen in a vertebrate subject is provided. In a specific embodiment, the method comprises the steps of providing an antigen-adjuvant composition comprising the antigen and a substantially non-toxic adjuvant molecule or molecules, and administering said antigen-adjuvant composition to the vertebrate subject, whereby an immune response is elicited. Preferably, the immune response comprises a systemic and/or mucosal immune response.
The IL-1xcex2 mutein adjuvant of the subject invention can be administered in the presence or absence of a vaccine antigen and either prior to or subsequent to vaccine antigen administration.